The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.

The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.

The aim of the theoretical part of this Master´s thesis is to make a research of possible ways in repairing PCC pavements without anchoring transverse joints. The pratical part is focused on the design of PCC pavements segmentations in experimental sections and evaluation measurement by testing muted impact.

No description available.

No description available.

<p>The practice of medicine has evolved from individual practice, mainly led by physicians, towards an interdisciplinary team-based activity that takes into consideration patients’ needs, preferences and values. This shift towards what is known as the patient-centered care (PCC) model requires better communication among medical staff and patients. Feedback is a key factor to improve team learning processes and also to understand patient perspectives. The objective of this thesis was to create a system to facilitate feedback processes within the Toronto General Hospital (TGH). To do so, a kiosk-based system was designed, allowing patients and health care providers to send feedback messages as well as positive recognition messages in appreciation of other people’s commitments and achievements. A pilot test was conducted by placing a kiosk in the General Internal Medicine unit at TGH for seven weeks. During this study, the application running the kiosk registered all user actions, with the objective of understanding how patients and staff interacted with the system. The kiosk was perceived as a positive, easy to use, and valuable tool by patients and providers. The different elements that need to be improved for future upgrades of the system are discussed. Overall, the use of kiosk-based systems to gather feedback should be considered when creating high performing teams in the health care domain.</p> / Master of Science (MSc)

Die Biosynthese von Chlorophyll ist komplex und umfasst mehr als ein Dutzend enzymatische Schritte. Es ist nur allzu selbstverständlich, dass eine Deregulation der Chlorophyllsynthese globale Effekte auf die Zelle hat. Um diese Konsequenzen näher zu beleuchten, wurden Arabidopsis thaliana Pflanzen mit chemisch induzierter Deaktivierung von zwei Chlorophyllbiosynthesegenen (CHLH bzw. CHL27) erzeugt sowie photoautotophe Zellsuspensionskulturen von Arabidopsis thaliana hinsichtlich kurzzeitig induzierter Signalprozesse untersucht. Die Resultate verdeutlichen, dass durch Fehlregulationen innerhalb der Chlorophyllbiosynthese erzeugte reaktive Sauerstoffspezies die Transkriptionskontrolle kernkodierter Gene beeinflussen. Die Untersuchung eines enzymatischen Schrittes der Chlorophyllbiosynthese trat in dieser Arbeit in den Hauptfokus: Die Bildung des fünften, isozyklischen Ringes im Chlorophyllmolekül, katalysiert durch das bisher unzureichend erforschte Enzym Mg-Protoporphyrin-IX-monomethylester-Cyclase (Cyclase). Anhand von transgenen Cyanobakterien und Pflanzen sollte das noch unbekannte Gen ycf54 hinsichtlich seiner physiologischen Funktion in dem Cyclase-Enzymschritt analysiert werden. Das Fehlen von Ycf54 in Synechocystis sp. PCC6803 bzw. des homologen LCAA-Proteins in Nicotiana tabacum und Arabidopsis thaliana führt zu starken Cyclase-Substrat-Akkumulationen, verringerten Chlorophyllgehalten und reduzierten Ycf59- bzw. CHL27-Proteingehalten. Ein Mangel von Ycf54/LCAA beeinträchtigt daher die Funktionalität des Cyclase-Komplexes und scheint sich zudem interessanterweise auch auf die Stabilität photosynthetischer Antennenkomplexe auszuwirken. Mittels Pulldown-Assays konnte für Arabidopsis thaliana die direkte physikalische Interaktion zwischen LCAA und CHL27 bestätigt werden. Darüber hinaus sind erste Hinweise für die Ferredoxin-NADP-Reduktase als potenziellen Interaktionspartner gezeigt. / Synthesis of chlorophyll is a complex metabolic process and encompasses more than a dozen enzymatic reactions. It is self-evident that a deregulation of chlorophyll biosynthesis evokes global cellular impacts. To elucidate these consequences Arabidopsis thaliana plants with chemically inducible deactivation of two chlorophyll biosynthesis genes (CHLH and CHL27, respectively) were generated and photoautotrophic cell suspension cultures of Arabidopsis thaliana were used for short induced signal processes. The results illustrate that reactive oxygen species provoked by a deregulated chlorophyll synthesis affect the control of transcription of nuclear genes. The investigation of one enzymatic step of chlorophyll biosynthesis was placed as main focus: The formation of the isocyclic ring of the chlorophyll molecule catalyzed by the Mg protoporphyrin IX monomethyl ester cyclase (short: cyclase), an enzyme which is not fully investigated so far. The still unknown hypothetical chloroplast open reading frame (ycf) ycf54 should be analyzed concerning it’s physiological function in the enzymatic step of the cyclase using transgenic cyanobacteria and plants. Lack of Ycf54 in Synechocystis sp. PCC6803 and the homologous LCAA protein in Nicotiana tabacum and Arabidopsis thaliana, respectively, leads to chlorophyll deficiency, a strong accumulation of the cyclase substrate and reduced protein contents of Ycf59 and CHL27, respectively. A deficit of Ycf54/LCAA impairs the functionality of the cyclase complex and also might compromise the stability of photosynthetic antenna complexes. Using pull-down assays a direct physical interaction between LCAA and CHL27 could be confirmed. Additionally, first evidences for ferredoxin NADP reductase as a potential interaction partner was given.

Since their inception, building materials are constantly improving. New ways to explore and improve the material's properties are constantly investigated (especially to improve the mechanical and deformation characteristics of the materials). This thesis is focused on identifying and comparing values of the modulus of elasticity of fine-grained composites. These features are compared with respect to water cement ratio, ageing and compressive strength of the investigated materials. Afterwards, the values of the modulus of elasticity are studied in the relation to the tested materials.

Depuis l'émergence de la pandémie de SARS-CoV-2 en décembre 2019, plus de 675 millions de cas confirmés ont été signalés dans le monde, dont 4,6 millions de cas au Canada uniquement. Bien que la plupart des individus récupèrent sans séquelles, 10 à 20 % des survivants signalent des symptômes persistants au-delà de quatre semaines après une infection par le SARS-CoV-2, tels que la fatigue, les altérations cognitives, la toux, l'anxiété, la dépression, la douleur thoracique et autres, connus sous le nom de COVID longue ou de condition post-SARS-CoV-2 (PCC). Par conséquent, la physiopathologie, le diagnostic et la prise en charge de la PCC sont devenus un axe de recherche majeur. Pour contribuer à la compréhension de la PCC, nous avons mené le projet IPCO (Institut de Recherches cliniques de Montréal (IRCM) Post-COVID-19 Research Clinic), en posant comme hypothèses 1 que les personnes infectés par le SARS-CoV-2 au Québec présenteraient des signes et symptômes fréquents et variés post-phase aiguë, affectant différents systèmes d'organes, et 2 Les niveaux élevés de D-dimères dans PCC ne sont pas pertinents pour les événements thromboemboliques 3 que Chez les individus atteints de la PCC, la vaccination contre la COVID-19 réduirait les symptômes de la PCC en diminuant l'inflammation. Pour évaluer ces hypothèses, nous avons recruté des participants âgés de plus de 18 ans, un à 18 mois après l'infection aiguë, présentant au moins un symptôme persistant, et programmé des visites de base et de suivi à 3-6 mois, 1 an et 2 ans post-infection aiguë. Chaque visite comprenait des évaluations cliniques, des prélèvements, des évaluations en laboratoire, des questionnaires sur l'alimentation et le bien-être, ainsi que des évaluations de la physiologie pulmonaire et cardiaque. Sur la base d'une étude allemande qui a catégorisé les symptômes du PCC et les individuals en trois groupes de sévérité, nous avons classé nos participants en trois niveaux de sévérité : non/légère (score du PCC <10,75), modérée (10,75 < score du PCC < 26,25) et sévère (score du PCC > 26,25). Cette thèse présente les résultats de trois sous-études IPCO. Dans l'étude descriptive, nous avons observé que la fatigue, les problèmes de mémoire et les maux de tête étaient les symptômes de PCC les plus courants, la majorité de nos participants étant des femmes et ayant été traités en ambulatoire pendant la phase aiguë. Dans l'étude transversale, nous avons constaté des différences significatives dans les mesures de santé et de bien-être à tous les moments, mais aucune différence significative dans les résultats des tests physiologiques entre les groupes PCC non/léger, modéré et sévère. Dans l'étude longitudinale, les marqueurs de l'inflammation se sont améliorés au fil du temps, mais le taux métabolique basal et la masse grasse ont augmenté. Dans la deuxième étude, nous avons observé une forte prévalence de participants ayant des niveaux de D-dimères, qui n'étaient pas associés à des événements thromboemboliques, et aucune corrélation entre le niveau de D-dimères et les niveaux de cytokines et de chimiokines. Dans la troisième étude, nous avons observé que les participants vaccinés présentaient significativement moins de symptômes de PCC. Notre étude fournit une meilleure compréhension de la physiopathologie du PCC et de l'effet de la vaccination sur le profil clinique et inflammatoire du PCC, ce qui pourrait aider à la conception d'outils de gestion clinique et de recherche futurs. / Since the emergence of the SARS-CoV-2 pandemic in December 2019, over 675 million confirmed cases have been reported globally, with 4.6 million cases in Canada alone. Although most individuals recover without residual disease, 10-20% of survivors report symptoms persisting beyond four weeks after SARS-CoV-2 infection, such as fatigue, cognitive impairments, cough, anxiety, depression, chest pain, and others known as long-COVID or post SARS-CoV-2 condition (PCC). Consequently, the pathophysiology, diagnosis, and management of PCC have become a significant focus of research. To contribute to the understanding of PCC, we conducted the IPCO (Institut de Recherches cliniques de Montréal (IRCM) Post-COVID-19 Research Clinic) project, hypothesizing that 1 SARS-CoV-2 infected individuals in Quebec would present frequent and varied signs and symptoms post-acute phase, affecting different organ systems, and that 2 high D-dimer level in PCC is irrelevant to thromboembolic events , and 3 in individuals with PCC, COVID-19 vaccination would decrease PCC symptoms by reducing inflammation. To evaluate these hypotheses, we enrolled participants aged >18 years, one to 18 months post-acute infection, with at least one persistent symptom, and scheduled baseline and follow-up visits at 3-6 months, 1 year, and 2 years post-acute infection. Each visit involved clinical evaluations, sampling, laboratory evaluations, diet and well-being questionnaires, and pulmonary and cardiac physiology evaluations. Based on a German study that categorized PCC symptoms and individuals into three severity groups, we classified our participants into three severity levels: non/mild (PCC score < 10.75), moderate (10.75 < PCC score < 26.25), and severe (PCC score > 26.25). This thesis reports the results of three IPCO studies. In the descriptive study, we observed that fatigue, memory problems, and headaches were the most common PCC symptoms, with the majority of our participants being female and managed as outpatients during the acute phase. In the cross-sectional study, we noted significant differences in health and well-being measurements at all time points, but no significant difference in physiological tests' results between different severity groups. In the longitudinal study, markers of inflammation improved over time, but the basal metabolic rate and body fat increased. In the second study, we observed a high prevalence of participants having D-dimer levels in blood, which were not associated with thromboembolic events, and no correlation between D-dimer levels and blood cytokine/ chemokine levels. In the third study, we observed that vaccinated participants had significantly fewer PCC symptoms, fewer organ systems affected, higher well-being scores, and lower blood cytokine/chemokine levels than the non-vaccinated group. We also observed correlations between certain cytokines/chemokines, as well as between clinical parameters and certain cytokines/chemokines. Our study provides a better understanding of the pathophysiology of PCC and effect of vaccination on the clinical and inflammatory profile of PCC, which could assist future research and clinical management tool design.

This dissertation presents a study of the molecular mechanism underlying the highly efficient solar energy conversion processes that occur in the Photosystem I (PS I) reaction centers in plants and bacteria. The primary electron donor P700 is at the heart of solar energy conversion process in PS I and the aim is to obtain a better understanding of the electronic and structural organization of P700 in the ground and excited states. Static Fourier Transform Infra-Red (FTIR) difference spectroscopy (DS) in combination with site directed mutagenesis and Density Functional Theory (DFT) based vibrational frequency simulations were used to investigate how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. (P700+-P700) FTIR DS at 77K were obtained from a series of mutants from the cyanobacterium Synechocystis sp. 6803 (S. 6803) where the amino acid residues near the C=O groups of the two chlorophylls of P700 where specifically changed. (P700+-P700) FTIR DS was also obtained for a set of mutants from C. reinhardtii where the axial ligand to A0-, the primary electron acceptor in PS I was modified. The FTIR DS obtained from these mutants provides information on the axial ligands, the hydrogen bonding status as well as the polarity of the environment of specific functional groups that are part of the chlorophyll molecules that constitute P700. Assignment of the FTIR bands to vibrational modes in specific types of environment is very difficult. In order to assist the assignment of the difference bands in experimental spectra DFT based vibrational mode frequency calculations were undertaken for Chl-a and Chl-a+ model molecular systems under different set of conditions; in the gas phase, in solvents using the Polarizable Continuum Model (PCM), in the presence of explicit solvent molecules using QM/MM methods, and in the presence of axial ligands and hydrogen bonds. DFT methods were also used to calculate the charge, spin and redox properties of Chl-a/Chl-a’ dimer models that are representative of P700, the primary electron donor in PS I.